PROJECT ABTRACT/SUMMARY The traditional Mediterranean diet has attracted increasing interest and has been shown to have beneficial effects on type 2 diabetes (T2D) risk factors. Recently, the PREDIMED (PREvencion con DIeta MEDiterranea) study was completed in Spain. This trial was a multicenter, randomized controlled trial in which 7,447 participants were randomly allocated to one of three nutritional interventions: a traditional Mediterranean diet supplemented with extra virgin olive oil; a traditional Mediterranean diet supplemented with tree nuts; or a control group receiving advice to follow a low-fat diet. Using T2D as a secondary outcome, it was found that compared to the control group, both intervention diets considered together significantly reduced the risk of T2D by 30%. Although the benefits of following a Mediterranean diet for T2D prevention are clearly observed in the PREDIMED trial, the biological mechanisms underlying these benefits are not completely understood. The proposed study will analyze data from archived fasting blood specimens assayed via state-of-the-art liquid chromatography tandem mass spectrometry technology. The study will employ a case-cohort study design with 251 incident cases of T2D and 694 randomly selected subcohort of participants free of diabetes at baseline as controls. We will assess the relationships between: i) urea cycle metabolites (arginine, ornithine, citrulline, methylarginine derivatives); ii) aromatic amino acids (histidine, phenylalanine, tyrosine); and iii) tryptophan metabolites (tryptophan, kynurenine, kynurenic acid, 3-hydroxyanthranilic acid, quinolinic acid) with T2D. We hypothesize that baseline and 1-year increase in arginine, ornithine, and citrulline, and decreases in methylarginine derivatives, aromatic amino acids, and decreases in tryptophan metabolites will be inversely associated with T2D risk. We further hypothesize that the directions of association for these metabolites with T2D will be similar for HOMA-IR and ?-cell function. This study poses important public health and clinical implications because urea metabolites, aromatic amino acids, and tryptophan metabolites may be suitable for direct diet and lifestyle interventions.